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Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses.
Pisanic, Nora; Antar, Annukka A R; Kruczynski, Kate L; Gregory Rivera, Magdielis; Dhakal, Santosh; Spicer, Kristoffer; Randad, Pranay R; Pekosz, Andrew; Klein, Sabra L; Betenbaugh, Michael J; Detrick, Barbara; Clarke, William; Thomas, David L; Manabe, Yukari C; Heaney, Christopher D.
  • Pisanic N; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA. Electronic address: npisani1@jhu.edu.
  • Antar AAR; Division of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
  • Kruczynski KL; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Gregory Rivera M; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Dhakal S; Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Spicer K; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Randad PR; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Pekosz A; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA; Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Klein SL; Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA; Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
  • Betenbaugh MJ; Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA.
  • Detrick B; Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
  • Clarke W; Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
  • Thomas DL; Division of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD, USA; Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
  • Manabe YC; Division of Infectious Diseases, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD, USA; Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
  • Heaney CD; Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA; Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA; Department of International Health, Bloomberg School of Pu
J Immunol Methods ; 514: 113440, 2023 03.
Article in English | MEDLINE | ID: covidwho-2234442
ABSTRACT

BACKGROUND:

Oral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance.

OBJECTIVES:

To optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays.

METHODS:

The salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December 2019 (n = 555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n = 398) and used to optimize and validate MIA performance (total n = 953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (µg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays.

RESULTS:

The sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 µg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se] = 100.0%; 95% confidence interval [CI] = 94.8%, 100.0%) and 108/109 negatives (specificity [Sp] = 99.1%; 95% CI = 97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se = 98.8%; 95% CI = 93.3%, 100.0%] and 127/127 negatives (Sp = 100%; 95% CI = 97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n = 30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N ρ = 0.76, RBD ρ = 0.83, S ρ = 0.82; all p < 0.001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N ρ = 0.68, RBD ρ = 0.78, S ρ = 0.79; all p < 0.001) and with plasma ELISA IgG (N ρ = 0.76, RBD ρ = 0.79, S ρ = 0.76; p < 0.001) were similar.

CONCLUSIONS:

A salivary SARS-CoV-2 IgG MIA produced consistently high Se (> 98.8%) and Sp (> 99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Blood Group Antigens / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study / Randomized controlled trials Topics: Long Covid / Vaccines / Variants Limits: Humans Language: English Journal: J Immunol Methods Year: 2023 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Blood Group Antigens / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study / Randomized controlled trials Topics: Long Covid / Vaccines / Variants Limits: Humans Language: English Journal: J Immunol Methods Year: 2023 Document Type: Article